1. Field of the Invention
This invention relates to a method and system for illuminating a sample and collecting scattered light from the sample. More particularly, the present invention relates to a method and system for illuminating an optically active sample and collecting scattered light from the sample in which a fiber optic bundle and a lens set is used for laser illumination and fluorescence collection.
2. Description of Related Art
Various techniques exist for separating particles, such as proteins, nucleic acids, and the like. For example, polyacrylamide gel electrophoresis separation can be used to separate two polypeptides of the same size but of different isoforms, or to separate polypeptides having very small differences in size. In addition, polyacrylamide gel electrophoresis can be used for DNA sequencing, in which nucleic acids are separated based on the size of DNA fragments.
In DNA sequencing, a thin gel is sandwiched between two plates (e.g., glass plates) having discrete lanes arrayed from one end of the glass plates to another. DNA fragments are introduced into the discrete lanes at one end of the plates. An electric field is then applied along the gel from either end of the plates, causing the DNA fragments to propagate through the gel from one end of the plates to the opposite end. The DNA fragments propagate in bands (or clumps), each having a discrete length measured by a number of nucleotides. Thus, for example, a band may be 10,000 nucleotides in length. The velocity of each band through the gel is dependent on the size (mass) of the DNA fragment and the charge on the fragment, with each band propagating at a different velocity. Consequently, each band in a lane passes a predetermined point along the lane at a discrete time.
The bands of DNA fragments may be detected using a variety of methods and associated apparatus. For example, as disclosed in U.S. Pat. No. 5,543,018, uncharged bands of DNA fragments can be detected by directing an incident beam of polarized light toward a predefined detection zone. The incident beam passes through the detection zone, resulting in an exiting beam, which is then analyzed to determine if its polarization differs from that of the incident beam. Differences in polarization between the incident beam and exiting beam are used to detect bands of DNA fragments.
In another detection technique, fluorescently labeled DNA fragments are illuminated by a narrow-band light source, focused into a small spot on the gel, at the wavelength that excites the fluorescent label. The labels within the illuminating spot, in turn, fluoresce light in an omnidirectional fashion that is shifted in wavelength from the illuminating spot. The fluoresced light is then collected and focused onto a light detector. The spot is repeatedly scanned across the gel in a direction perpendicular to the electric field. This scanning builds an image of the bands in the gel, because the bands are propagating along the direction of the electric field. The maximum resolution of the resultant image is determined by the size of the illuminating spot, for example, 50 .mu.m.
Currently, a confocal system is used for scanning the spot across the gel. FIG. 1 shows a typical confocal system 100, in which the same lens 102 is used to focus an illuminating spot 104 into a gel 106 and to collect fluoresced light emitted by a sample (not shown) within the gel 106. The illuminating beam 110 is produced by a laser diode 112 and is then collimated by an aspherical lens 114. An interference filter 116 is used to reject laser light from the beam 110 that is within the spectrum of the fluoresced light. The filtered beam 110 then propagates to a dichroic mirror 118 that reflects the beam 110 at a 90 degree angle. The reflected beam 110 is directed to a moving mirror and lens set 102, which moves along the beam 110, thus scanning the focused spot 104 across the gel 106, which is supported between two glass plates 108. The lens 102 is diffraction limited and has a large numerical aperture so that a significant fraction of fluoresced light emitted by the sample is collected and formed into a well collimated beam 108 of fluoresced light. The fluoresced light beam 108 propagates back along the path of the illuminating beam 110 to the dichroic mirror 118. The mirror 118 is selected so that the fluoresced light beam 108 is transmitted through the mirror 118, propagating through a filter 122 that rejects all light outside the spectra of the fluoresced light 112. A lens 124 then focuses the fluoresced light beam 120 onto an avalanche photodiode detector and amplifier 126.
Although confocal system 100 is efficient and relatively simple, its suffers a significant drawback in that it is difficult to align. Alignment of the parts comprising system 100 is completely interdependent, because an adjustment to any part of the system 100 requires that all other parts of the system be adjusted as well. This causes significant problems in mass manufacturing applications. Moreover, confocal systems require a stable mechanical system to maintain the system in alignment. Further, the confocal system incorporates a number of optical parts, including the dichroic mirror 118, and has a relatively complex structure.
Therefore, a need exists for a method and apparatus for detecting optically active molecules, such as charged bands of DNA fragments, in which alignment is simple and efficient and the structure is less complex and smaller than confocal systems. The present invention provides such a method and apparatus.